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Currently, the genus Arctia Schrank, 1802 includes approximately 16 species in the Palaearctic region, depending on the taxonomic interpretation. Here, populations of the Arctia villica (Linnaeus, 1758) morphospecies complex were studied from Europe to the Middle East (Turkey, northern Iran) by molecular methods. Morphological treatment has traditionally revealed the presence of five nominal taxa: A. villica (Linnaeus, 1758), A. angelica (Boisduval, 1829), A. konewkaii (Freyer, 1831), A. marchandi de Freina, 1983, and A. confluens Romanoff, 1884. The molecular approach tests whether they represent well-delimited species. Subsequently, this study corroborates the suitability of the mitochondrial cytochrome c oxidase subunit 1 (COI) marker sequence for species delimitation. In total, 55 barcodes of the Arctia villica complex were compared, and two molecular species delimitation algorithms were applied to reveal the potential Molecular Operational Taxonomic Units (MOTUs), namely the distance-based Barcode Index Number (BIN) System, and the hierarchical clustering algorithm based on a pairwise genetic distances approach using the Assemble Species by Automatic Partitioning (ASAP). The applied ASAP distance-based species delimitation method for the analysed dataset revealed an interspecific threshold of 2.0–3.5% K2P distance as suitable for species identification purposes of the Iberian A. angelica and the Sicilian A. konewkaii and less than 2% for the three taxa of the A. villica clade: A. villica, A. confluens, and A. marchandi. This study contributes to a better understanding of the taxonomy of the genus Arctia and challenges future revision of this genus in Turkey, the Caucasus, Transcaucasia as well as northern Iran using standard molecular markers.

The systematics of the Old World Spiranthes sinensis (Pers.) Ames species complex (Orchidaceae) has been complicated by its wide distribution and morphological variations. Within the species complex, S. australis Lindl. has been generally accepted as the only Spiranthes Rich. species distributed on the Japanese mainland. The present study provides morphological, phylogenetic, and ecological evidence for the recognition of S. hachijoensis Suetsugu as a new species of the S. sinensis species complex on the Japanese mainland. Spiranthes hachijoensis is morphologically similar to S. hongkongensis S.Y. Hu & Barretto and S. nivea T.P. Lin & W.M. Lin, sharing a degenerated rostellum, pollinia without a viscidium, and distinctly trilobed stigma. However, the taxon can be morphologically distinguished from S. hongkongensis by its glabrous rachis, ovaries, and sepals, and from S. nivea by its papillate labellum disc, larger papillate basal labellum callosities, and glabrous rachis, ovaries, and sepals. The autogamy and flowering phenology (i.e., earlier flowering) of S. hachijoensis are most likely responsible for premating isolation from the sympatric S. australis. A MIG-seq-based high-throughput molecular analysis indicated that the genetic difference between S. hachijoensis and its putative sister species S. sinensis is comparable to, or even greater than, the genetic difference between pairs of other species within the S. sinensis species complex. Our multifaceted approach strongly supports the recognition of S. hachijoensis as a morphologically, phenologically, phylogenetically, and ecologically distinct species.

The study of a well-preserved collection of discodorid nudibranchs collected in Koumac, New Caledonia, revealed the presence of seven species new to science belonging to the genera Atagema, Jorunna, Rostanga, and Sclerodoris, although some of the generic assignments are tentative as the phylogeny of Discodorididae remains unresolved. Moreover, a poorly known species of Atagema originally described from New Caledonia is re-described and the presence of Sclerodoris tuberculata in New Caledonia is confirmed with molecular data. All the species described herein are highly cryptic on their food source and in the context of the present study the term “cryptic” is used to denote such species. This paper highlights the importance of comprehensive collecting efforts to identify and document well-camouflaged taxa.

The present study investigated the ability of DNA barcoding to reliably identify the endemic freshwater species in Turkey, known as biodiversity hotspots. The barcode region (652 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) was used to barcode 153 individuals from 13 morphologically identified species of the genus Alburnoides. Based on the Kimura two-parameter (K2P) evolution model, the average interspecific distance (0.0595) was 31-fold higher than the average intraspecific distance (0.0019). There was a clear-cut barcode gap (0.0158–0.0187) between maximum intraspecific distance (A. tzanevi and A. velioglui) and minimum nearest-neighbour distance (A. freyhofi and A. kurui) for Anatolian Alburnoides species and a common genetic threshold of 0.0158 sequence divergence was defined for species delimitation. The multiple species delimitation methods (ABGD, ASAP, GMYC and bPTP) revealed a total of 11 molecular operational taxonomic units (MOTUs) for 13 morphospecies. Neighbour-joining (NJ), Maximum Likelihood (ML) and Bayesian Inference (BI) tree analysis indicated that all haplotypes were clustered into two major clades, which corresponded to eleven Alburnoides species clusters, with strong bootstrap support. Furthermore, all the specimens clustered in concurrence with the morpho-taxonomic status of the species, except for two species (A. coskuncelebii and A. emineae) that were morphologically differentiated, but showed overlap in variation for COI-based DNA barcode data with other species. Overall, present results identified that COI-based DNA barcoding is effective for species identification and cataloguing of genus Alburnoides in Turkey.

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.

Barcode libraries are generally assembled with two main objectives in mind: specimen identification and species discovery/delimitation. In this study, the standard COI barcode region was sequenced from 681 specimens belonging to katydids (Tettigoniidae), cave crickets (Rhaphidophoridae), and leaf-rolling crickets (Gryllacrididae) from Zhejiang Province, China. Of these, four COI-5P sequences were excluded from subsequent analyses because they were likely NUMTs (nuclear mitochondrial pseudogenes). The final dataset consisted of 677 barcode sequences representing 90 putative species-level taxa. Automated cluster delineation using the Barcode of Life Data System (BOLD) revealed 118 BINs (Barcodes Index Numbers). Among these 90 species-level taxa, 68 corresponded with morphospecies, while the remaining 22 were identified based on reverse taxonomy using BIN assignment. Thirteen of these morphospecies were represented by a single barcode (so-called singletons), and each of 19 morphospecies were split into more than one BIN. The consensus delimitation scheme yielded 55 Molecular Operational Taxonomic Units (MOTUs). Only four morphospecies (Imax > DNN) failed to be recovered as monophyletic clades (i.e., Elimaea terminalis, Phyllomimus klapperichi, Sinochlora szechwanensis and Xizicus howardi), so it is speculated that these may be species complexes. Therefore, the diversity of katydids, cave crickets, and leaf-rolling crickets in Zhejiang Province is probably slightly higher than what current taxonomy would suggest.

The use of DNA has helped to improve and speed up species identification and delimitation. However, it also provides new challenges to taxonomists. Incongruence of outcome from various markers and delimitation methods, bias from sampling and skewed species distribution, implemented models, and the choice of methods/ priors may mislead results and also may, in conclusion, increase elements of subjectivity in species taxonomy. Lack of direct diagnostic outcome from most contemporary molecular delimitation approaches and need of reference to existing and best sampled trait reference systems, reveals the need of refining criteria of species diagnosis and diagnosability in the current framework of nomenclature codes and good practices to avoid nomenclatorial instability, parallel taxonomies, and consequently more and new taxonomic impediment.

High altitude ecosystems are often home to endemic species that have evolved in isolation from their low elevation counterparts. In many cases, especially in insects, such mountain endemics are often apterous (due to their reduced ability to fly and disperse). In most cases, so far, these mountain endemics are genetically differentiated from lowland sister species or populations. During an excursion in Central Spain, we encountered two such strikingly different morphotypes of the rove beetle Lobrathium multipunctum (Gravenhorst, 1802) (Coleoptera, Staphylinidae, Paederinae). The morphotype from high elevation was smaller and paler than those at low elevation, which were slightly larger, darker and regularly coloured. The high altitude morphotype was earlier considered a separate species (Lobrathium hispanicum Dodero, 1916) from its widespread lower-land counterpart (Lobrathium multipunctum (Gravenhorst, 1802)) before their relatively recent synonymy. Using the cytochrome c oxidase subunit I (COI) barcode region, we tested whether these distinct morphotypes are distinct species. We found that their synonymy is supported, based on multiple species delimitation methods. We suggest that this phenomenon may be more widespread amongst insects and other organismal groups. We note that the presence of high altitude morphotypes which are phylogenetically nested within, or genetically identical to, widespread lowland species (regular morphotype) is rarely reported in the literature on beetles (and other insects). These findings thus highlight the need for caution when describing mountain endemics and further highlight DNA barcoding as a helpful tool for their study.

Molecular tools have been employed to improve the knowledge about freshwater Neotropical fishes. Such approaches supporting studies of groups including species complexes such as Astyanax, one of the most diversified and taxonomically complex genus of the family Characidae. Here, we employed species delimitation analyses in four Astyanax species described for the upper Paraguaçu River basin, a drainage within Northeastern Mata Atlântica freshwater ecoregion with high endemism. We implemented single and multilocus approaches based on two mitochondrial and one nuclear markers. Cytochrome c Oxidase I sequences previously available for Astyanax species were also added to our dataset. The single locus analyses showed A. epiagos, A. rupestris, and A. aff. rupestris as different Molecular Operational Taxonomic Units (MOTUs), while A. brucutu and A. lorien were grouped. However, the multilocus approach distinguished these two species and showed congruence for the remaining single locus results. Astyanax aff. rupestris was separated into two MOTUs using both approaches, highlighting the need for an integrative taxonomic revision including A. aff. rupestris. These findings contribute to a better understanding of the diversity of this fish group in the upper Paraguaçu, identifying hidden diversity and reinforcing the relevance of this hydrographic system as a notable hotspot for ichthyofauna biodiversity endemism. Resumo Ferramentas moleculares têm sido empregadas para melhorar o conhecimento sobre os peixes Neotropicais. Tais abordagens apoiam estudos de grupos que incluem complexos de espécies, como Astyanax, um dos gêneros mais diversificados e taxonomicamente complexos dentro da família Characidae. Neste estudo, nós empregamos análises de delimitação de espécies em quatro espécies de Astyanax recentemente descritas da bacia do alto rio Paraguaçu, uma drenagem dentro da ecorregião Mata Atlântica Nordeste que apresenta alto endemismo. Nós realizamos abordagens de loco único e multilocos baseadas em dois marcadores mitocondriais e um nuclear. Sequências de Citocromo c Oxidase I anteriormente disponíveis para espécies de Astyanax foram adicionadas ao nosso conjunto de dados. As análises de loco único mostraram A. epiagos, A. rupestris e A. aff. rupestris como diferentes Unidades Taxonômicas Operacionais Moleculares (MOTUs), enquanto A. brucutu e A. lorien foram agrupadas. Entretanto, a abordagem multilocos distinguiu estas duas espécies e mostrou congruência com os demais resultados das análises de loco único. Astyanax aff. rupestris foi separada em duas MOTUs usando ambas as abordagens, sugerindo a necessidade de uma revisão taxonômica integrativa incluindo A. rupestris e ambas A. aff. rupestris. Esses achados contribuem para uma melhor compreensão da diversidade desse grupo de peixes na bacia do rio Paraguaçu, identificando diversidade oculta e reforçando a relevância desse sistema hidrográfico como um notável hotspot de endemismo da biodiversidade da ictiofauna.

To date, a rather large set of both mathematical theories for species delimitation, based on single-locus genetic data, and their implementations as software products, has been accumulated. Comparison of the efficiencies of different delineation methods in the task of accumulating and analyzing data with reference to different taxa in different regions, is vital. The aim of this study was to compare the efficiency of fifteen single-locus species delimitation methods using the example of a fish species found in a single lake in European Russia (Lake Plescheyevo) with reference to other sequences of revealed taxa deposited in international databases. We analyzed 186 original COI sequences belonging to 24 haplotypes, and 101 other sequences previously deposited in GenBank and BOLD. Comparison of all 15 alternative taxonomies demonstrated that all methods adequately separate only the genera, while the number of delimited mOTUs differed from 16 (locMin) to 43 (HwM/CoMa). We can assume that the effectiveness of each method is correlated with the number of matches based on Ctax and MatchRatio criteria. The most comparable results were provided by bGMYC, mPTP, STACEY, KoT and ASAP and the most synchronous results were obtained from bGMYC, mPTP, STACEY and ASAP. We believe that these results are maximally realistic in the number of revealed mOTUs. A high genetic diversity, resulting in the existence of several mOTUs and phylogenetic lineages within many species, demonstrates the usefulness of the “polymorphic species” concept, which does not underestimate species richness and does not prevent the rational use and protection of biodiversity.

Species of Cystolepiota are known as diminutive lepiotaceous fungi with a worldwide distribution. Previous studies revealed that Cystolepiota is not monophyletic and preliminary DNA sequence data from recent collections suggested that several new species exist. Based on multi-locus DNA sequence data (the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2, ITS; the D1–D2 domains of nuc 28S rDNA, LSU; the most variable region of the second-largest subunit of RNA polymerase II, rpb2 and a portion of the translation–elongation factor 1-α. tef1), C. sect. Pulverolepiota forms a distinct clade separating from Cystolepiota. Therefore, the genus Pulverolepiota was resurrected and two combinations, P. oliveirae and P. petasiformis were proposed. With the integration of morphological characteristics, multi-locus phylogeny, and information on geography and habitat, two new species, viz. C. pseudoseminuda and C. pyramidosquamulosa, are described and C. seminuda was revealed to be a species complex containing at least three species, viz. C. seminuda, C. pseudoseminuda, and Melanophyllum eryei. In addition, C. seminuda was re-circumscribed and neo-typified based on recent collections.

Documenting species and population diversity is becoming increasingly important as the destruction and degradation of natural ecosystems are leading to a worldwide biodiversity loss. Despite the rapid development of genetic tools, many species remain undocumented and little is known about the diversity of individuals and populations, especially for tropical African plants. In this study, we aim to identify putative hidden species and/or differentiated populations in the tropical African tree Staudtia kamerunensis Warb. (Myristicaceae), a widespread species characterized by a high morphological diversity and a complex taxonomical history. Historical herbarium vouchers were sampled and leaf or cambium samples were collected in the field, dried in silica gel, and subsequently genotyped at 14 microsatellite loci (SSRs), as well as sequenced for two nuclear genes (At103, Agt1) and one plastid region (psbA-trnH). These genetic data were then analyzed using Bayesian clustering, population genetics, and the construction of haplowebs to assess genetic clustering patterns, the distribution of genetic diversity, and genetic differentiation among populations. Multiple genetically differentiated clusters were observed in parapatry throughout Central Africa. Genetic diversity was high and similar among these clusters, apart from the most differentiated populations in southeast Democratic Republic of the Congo (DR Congo), which showed lower levels of genetic diversity. The genetic breaks detected between S. kamerunensis populations are likely not indicative of hidden species but rather result from ancient rainforest fragmentation during cold and dry periods in the Pliocene and/or Pleistocene. The strong genetic divergence between populations in southeast DR Congo could be the result of an ongoing speciation linked to ecological niche differentiation.

Discovery of cryptic diversity is essential to understanding both the process of speciation and the conservation of species. Determining species boundaries in fern lineages represents a major challenge due to lack of morphologically diagnostic characters and frequent hybridization. Genomic data has substantially enhanced our understanding of the speciation process, increased the resolution of species delimitation studies, and led to the discovery of cryptic diversity. Here, we employed restriction-site-associated DNA sequencing (RAD-seq) and integrated phylogenomic and population genomic analyses to investigate phylogenetic relationships and evolutionary history of 16 tree ferns with marginate scales (Cyatheaceae) from China and Vietnam. We conducted multiple species delimitation analyses using the multispecies coalescent (MSC) model and novel approaches based on genealogical divergence index (gdi) and isolation by distance (IBD). In addition, we inferred species trees using concatenation and several coalescent-based methods, and assessed hybridization patterns and rate of gene flow across the phylogeny. We obtained highly supported and generally congruent phylogenies inferred from concatenated and summary-coalescent methods, and the monophyly of all currently recognized species were strongly supported. Our results revealed substantial evidence of cryptic diversity in three widely distributed Gymnosphaera species, each of which was composite of two highly structure lineages that may correspond to cryptic species. We found that hybridization was fairly common between not only closely related species, but also distantly related species. Collectively, it appears that scaly tree ferns may contain cryptic diversity and hybridization has played an important role throughout the evolutionary history of this group.

Parapatrically distributed taxa pose a challenge for species delimitation due to the presence of gene flow and inherent arbitrariness of exactly defining the species boundaries in such systems. We tackled the problem of species delimitation in a parapatric species pair of Melitaea butterflies using two popular genomic methods—double digest restriction-site associated DNA sequencing (ddRAD) and target enrichment. We compared newly generated target enrichment dataset with 1733 loci to the already available ddRAD data from a previous study on the same set of specimens using a suite of phylogenetic, population genetic, and species delimitation methods. We recovered consistent phylogenetic relationships across the datasets, both demonstrating the presence of a genetically distinct Balkan lineage and paraphyly of Melitaea athalia with respect to Melitaea celadussa. Population genetic STRUCTURE analyses supported the presence of two species when using ddRAD data, but three species when using target enrichment, while a Bayes factor delimitation analysis found both two and three species scenarios equally decisive in both datasets. As the results obtained from both methods were largely congruent, we discuss some practical considerations and benefits of target enrichment over RAD sequencing. We conclude that the choice of method of genomic data collection does not influence the results of phylogenetic analyses at alpha taxonomic level, given a sufficient number of loci. Finally, we recommend a solution for delineating species in parapatric scenarios by proposing that parapatric taxa be consistently classified as subspecies or complete species, but not both, to promote taxonomic stability., This work was supported by Academy of Finland (grant no. 314702) to Marko Mutanen. Roger Vila was supported by Grant PID2019-107078GB-I00 funded by MCIN/AEI/ 10.13039/501100011033. Vlad Dincă was supported by the Academy of Finland (Academy Research Fellow, decision no. 328895).

Ulva is a green macroalgal genus with rich species diversity and worldwide distribution. While current knowledge on Ulva diversity focuses on temperate regions, genetic and morphological data in tropical and subtropical areas are scarce and the species richness is not clearly defined. The genus is known for its bloom-forming ability that can induce green tides leading to severe environmental and economic damage. In the last two decades, several important blooms of Ulva spp. have occurred in New Caledonia, requiring further investigations to identify the species involved. As knowledge of New Caledonian Ulva diversity is limited, an update to the Ulva spp. inventory in the area is essential. Based on Ulva specimens collected throughout New Caledonia (Grande Terre, Isle of Pines and Loyalty Islands), we (1) reassessed species diversity using species delimitation methods, (2) analysed morpho-anatomical characters to identify species and/or enrich their diagnosis, and (3) reconstructed a multilocus phylogeny (ITS, rbcL, tufA) of the genus. We found 21 secondary species hypotheses (SSHs) among our dataset, from which five were successfully assigned to U. lactuca, U. ohnoi, U. tepida, U. meridionalis and U. taeniata. Ten SSHs were defined as new species for which we provided taxonomic description, and six other SSHs were singletons that will need to be data-enriched for better interpretation. Our concatenated multilocus matrix included 61 Ulva species. Of these, 15 species were found in New Caledonia and were moderately to strongly supported. Among the Ulva species found in New Caledonia, seven are known to be bloom-forming which highlights the need for strict regulation and regular monitoring of water quality, particularly in areas exposed to strong nutrient input where these species can form green tides. Highlights Ulva diversity in New Caledonia was reassessed with 15 species highlighted. Ten new species have been discovered in New Caledonia. Indigenous species caused recent green tides in New Caledonia.

The genus Morchella has gone through turbulent taxonomic treatments. Although significant progress in Morchella systematics has been achieved in the past decade, several problems remain unresolved and taxonomy in the genus is still in flux. In late 2019, a paper published in the open-access journal Scientific Reports raised serious concerns about the taxonomic stability of the genus, but also about the future of academic publishing. The paper, entitled “High diversity of Morchella and a novel lineage of the esculenta clade from the north Qinling Mountains revealed by GCPSR-based study” by Phanpadith and colleagues, suffered from gross methodological errors, included false results and artifactual phylogenies, had misapplied citations throughout, and proposed a new species name invalidly. Although the paper was eventually retracted by Scientific Reports in 2021, the fact that such an overtly flawed and scientifically unsound paper was published in a high-ranked Q1 journal raises alarming questions about quality controls and safekeeping procedures in scholarly publishing. Using this paper as a case study, we provide a critical review on the pitfalls of Morchella systematics followed by a series of recommendations for the delimitation of species, description of taxa, and ultimately for a sustainable taxonomy in Morchella. Problems and loopholes in the academic publishing system are also identified and discussed, and additional quality controls in the pre- and post-publication stages are proposed.

Species identities of Goniobranchus nudibranchs with white bodies and various marginal bands have long been problematic. In this study, specimens of these Goniobranchus nudibranchs from the Philippines, Peninsular Malaysia, Japan, Papua New Guinea, and Madagascar were analyzed and molecular data were obtained in order to re-examine the relationships between species within this “white Goniobranchus with marginal bands” group. The analyses clearly recovered six species groups corresponding to the described species Goniobranchus albonares, G. preciosus, G. rubrocornutus, G. sinensis, and G. verrieri as well as one new species, G. fabulus Soong & Gosliner, sp. nov. Notably, G. preciosus, G. sinensis, G. rubrocornutus, G. verrieri, and G. fabulus Soong & Gosliner, sp. nov. exhibit color variation and polymorphism, suggesting that some aspects of color patterns (e.g., presence or absence of dorsal spots) may not always be useful in the identification of species in the “white Goniobranchus with marginal bands” group, whereas other features such as gill and rhinophore colors and the arrangement and colors of the mantle marginal bands are more diagnostic for each species.

The leafcutting bees of the leachella group of Megachile Latreille subgenus Eutricharaea Thomson are revised for the Western Palaearctic region using a combination of morphology and phylogenetic analyses of three genes (COI, LW-Rhodopsin, CAD). Although only seven species are recognized, much effort was needed to link delimitated taxonomic units to taxon names because of the difficulties in identifying type specimens. Numerous types were in a poor state of conservation, preventing straight-forward identification using morphology. This was in some cases aggravated by the fact that they often belonged to a sex that could not easily be identified; one type was a gynandromorph specimen whose identification is even more challenging. In several cases, the type locality was vague or unclear; in three cases, the type specimens originated from introduced populations for which the source of the introduction needed to be determined using DNA barcoding. In two cases, the type specimens consisted of several body parts not originating from a single individual but from two heterospecific specimens. We argue that this tedious nomenclatural work would have been greatly facilitated if a reference library of type specimens had been available. Our revision leads to the following taxonomic changes. Megachile argentata (Fabricius, 1793), described from northern Africa and with a convoluted taxonomic history, is demonstrated, based on morphometric analyses of its lectotype, to be conspecific with the species hitherto known as M. pilidens Alfken, 1924. After discussing and excluding several alternative options that would minimize nomenclatural changes, we place M. pilidens in synonymy with M. argentata (syn. nov.). Two new subspecies are described for morphologically slightly divergent insular populations, M. leachella cretica Praz, ssp. nov. from Crete, and M. leachella densipunctata Praz, ssp. nov. from Cyprus. In addition, M. albipila Pérez, 1895 is treated as a subspecies of M. leachella Curtis, 1828 (stat. nov.). The following new synonymies are proposed: M. compacta Pérez, 1895 (not M. compacta Smith, 1879) and the replacement name M. crassula Pérez, 1896, M. argyrea Cockerell, 1931 and Perezia maura Ferton, 1914, are placed in synonymy with M. argentata (syn. nov.). M. beaumonti Benoist, 1951, is newly treated as a valid species (stat. rev.). M. schmiedeknechti Costa, 1884 is treated as a subspecies of M. argentata (stat. nov.), and M. xanthopyga Pérez, 1895 is placed in synonymy with M. argentata schmiedeknechti (syn. nov.). M. bioculata Pérez, 1902, M. discriminata Rebmann, 1968 and M. ichnusae Rebmann, 1968 are placed in synonymy with M. leachella (syn. nov.). M. variscopa Pérez, 1895, M. timberlakei Cockerell, 1920, M. atratula Rebmann, 1968, M. striatella Rebmann, 1968 and M. sudai Ikudome, 1999 are placed in synonymy with M. pusilla Pérez, 1894. Lectotypes are designated for M. albipila, M. bioculata, M. compacta Pérez, M. pusilla, M. variscopa and M. xanthopyga.

In this study the aim was to resolve the taxonomy of several species of Argyria Hübner (Pyraloidea, Crambinae) with previously unrecognised morphological variation. By analysing the DNA barcode (COI-5P) in numerous specimens, the aim was to reconstruct phylogenetic relationships between species, to provide better evidence for synonymies, and to circumscribe their geographical distribution. Using an innovative DNA hybridisation capture protocol, the DNA barcode of the lectotype of Argyria lacteella (Fabricius, 1794) was partially recovered for comparison with the 229 DNA barcode sequences of Argyria specimens available in the Barcode of Life Datasystems, and this firmly establishes the identity of the species. The same protocol was used for the following type specimens: the Argyria abronalis (Walker, 1859) holotype, thus confirming the synonymy of this name with A. lacteella, the holotype of A. lusella (Zeller, 1863), syn. rev., the holotype of A. multifacta Dyar, 1914, syn. nov. newly synonymised with A. lacteella, and a specimen of Argyria diplomochalis Dyar, 1913, collected in 1992. In addition, nine specimens of A. lacteella, A. diplomochalis, A. centrifugens Dyar, 1914 and A. gonogramma Dyar, 1915, from North to South America were sampled using classical COI amplification and Sanger sequencing. Argyria gonogramma Dyar, described from Bermuda, is the name to be applied to the more widespread North American species formerly identified as A. lacteella. Following morphological study of its holotype, Argyria vestalis Butler, 1878, syn. nov. is also synonymised with A. lacteella. The name A. pusillalis Hübner, 1818, is considered a nomen dubium associated with A. gonogramma. The adult morphology is diagnosed and illustrated, and distributions are plotted for A. lacteella, A. diplomochalis, A. centrifugens, and A. gonogramma based on slightly more than 800 specimens. For the first time, DNA barcode sequences are provided for the Antillean A. diplomochalis. This work provides a modified, improved protocol for the efficient hybrid capture enrichment of DNA barcodes from 18th and 19th century type specimens in order to solve taxonomic issues in Lepidoptera.

Nearly 90% of fungal diversity, one of the most speciose branches in the tree of life, remains undescribed. Lichenized fungi as symbiotic associations are still a challenge for species delimitation, and current species diversity is vastly underestimated. The ongoing democratization of Next-Generation Sequencing is turning the tables. Particularly, reference-based RADseq allows for metagenomic filtering of the symbiont sequence and yields robust phylogenomic trees of closely related species. We implemented reference-based RADseq to disentangle the evolution of neuropogonoid lichens, which inhabit harsh environments and belong to Usnea (Parmeliaceae, Ascomycota), one of the most taxonomically intriguing genera within lichenized fungi. Full taxon coverage of neuropogonoid lichens was sampled for the first time, coupled with phenotype characterizations. More than 20,000 loci of 126 specimens were analyzed through concatenated and coalescent-based methods, including time calibrations. Our analysis addressed the major taxonomic discussions over recent decades. Subsequently, two species are newly described, namely U. aymondiana and U. fibriloides, and three species names are resurrected. The late Miocene and Pliocene-Pleistocene boundary is inferred as the timeframe for neuropogonoid lichen diversification. Ultimately, this study helped fill the gap of fungal diversity by setting a solid backbone phylogeny which raises new questions about which factors may trigger complex evolutionary scenarios.

21 pages, 4 figures, 1 table, supporting information https://doi.org/10.1002/ece3.9740.-- Data Availability Statement: Raw RNA-seq data and mt-mutS sequences have been uploaded to NCBI's SRA (BioProject ID: PRJNA847883) and GenBank databases (Accessions numbers: ON804207-ON804214), respectively. Final sequence alignments and microsatellite data are available on DRYAD (https://doi.org/10.5061/dryad.r7sqv9sfm), The accurate delimitation of species boundaries in nonbilaterian marine taxa is notoriously difficult, with consequences for many studies in ecology and evolution. Anthozoans are a diverse group of key structural organisms worldwide, but the lack of reliable morphological characters and informative genetic markers hampers our ability to understand species diversification. We investigated population differentiation and species limits in Atlantic (Iberian Peninsula) and Mediterranean lineages of the octocoral genus Paramuricea previously identified as P. clavata. We used a diverse set of molecular markers (microsatellites, RNA-seq derived single-copy orthologues [SCO] and mt-mutS [mitochondrial barcode]) at 49 locations. Clear segregation of Atlantic and Mediterranean lineages was found with all markers. Species-tree estimations based on SCO strongly supported these two clades as distinct, recently diverged sister species with incomplete lineage sorting, P. cf. grayi and P. clavata, respectively. Furthermore, a second putative (or ongoing) speciation event was detected in the Atlantic between two P. cf. grayi color morphotypes (yellow and purple) using SCO and supported by microsatellites. While segregating P. cf. grayi lineages showed considerable geographic structure, dominating circalittoral communities in southern (yellow) and western (purple) Portugal, their occurrence in sympatry at some localities suggests a degree of reproductive isolation. Overall, our results show that previous molecular and morphological studies have underestimated species diversity in Paramuricea occurring in the Iberian Peninsula, which has important implications for conservation planning. Finally, our findings validate the usefulness of phylotranscriptomics for resolving evolutionary relationships in octocorals, This work was funded by program BIOMARES, a Pew Marine Fellowship, and FCT – Foundation for Science and Technology UIDB/04326/2020, UIDP/04326/2020, LA/P/0101/2020, PTDC/BIA-CBI/6515/2020; EU-BiodivERsA BiodivRestore-253 (FCT: DivRestore/0013/2020); EU Horizon 2020 research and innovation program (MERCES: Grant no. 689518); as well as the Inaqua Conservation Fund (Oceanário de Lisboa and National Geographic Channel) and National Geographic Society/Waitt Grant no. W153-11. This article is also a product of the French network on Marine Connectivity (GDR MarCo). M.C. was supported by postdoctoral fellowships of projects HABMAR (Grant no. MAR-01.04.02-FEAMP-0018) co-financed by the European Maritime and Fisheries Fund of the Operational Program MAR 2020 for Portugal (Portugal 2020), and BiodivAMP (Grant no. FA_06_2017_045) financed by the Directorate-General for Sea Policy of the Ministry of Economy and Sea for Portugal under the Operational Program Fundo Azul. JBL was funded by assistant researcher contract framework of the RD Unit—UID/Multi/04423/2019 – Interdisciplinary Centre of Marine and Environmental Research—financed by the European Regional Development Fund (ERDF) through COMPETE2020—Operational Program for Competitiveness and Internationalization (POCI) and national funds through FCT/MCTES (PIDDAC). JB was funded by a FCT PhD scholarship SFRH/BD/72501/2010, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Fossil spinicaudatan taxonomy heavily relies on carapace features (size, shape, ornamentation) and palaeontologists have greatly refined methods to study and describe carapace variability. Whether carapace features alone are sufficient for distinguishing between species of a single genus has remained untested. In our study, we tested common palaeontological methods on 481 individuals of the extant Australian genus Ozestheria that have been previously assigned to ten species based on genetic analysis. All species are morphologically distinct based on geometric morphometrics (p ≤ 0.001), but they occupy overlapping regions in Ozestheria morphospace. Linear discriminant analysis of Fourier shape coefficients reaches a mean model performance of 93.8% correctly classified individuals over all possible 45 pairwise species comparisons. This can be further increased by combining the size and shape datasets. Nine of the ten examined species are clearly sexually dimorphic but male and female morphologies strongly overlap within species with little influence on model performance. Ornamentation is commonly species‐diagnostic; seven ornamentation types are distinguished of which six are species‐specific while one is shared by four species. A transformation of main ornamental features (e.g. from punctate to smooth) can occur among closely related species suggesting short evolutionary timescales. Our overall results support the taxonomic value of carapace features, which should also receive greater attention in the taxonomy of extant species. The extensive variation in carapace shape and ornamentation is noteworthy and several species would probably have been assigned to different genera or families if these had been fossils, bearing implications for the systematics of fossil Spinicaudata., Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659, https://doi.org/10.5061/dryad.fbg79cnxs

Mordellistena A. Costa, 1854, the most species-rich genus of tumbling flower beetles comprises more than 800 species worldwide and more than 150 reported from Europe. Here, a new species Mordellistena (s. str.) platypoda is described from the island of Ischia in Italy. The species hypothesis is based primarily on morphological characters which are visualised using scanning electron microscopy images, high-resolution photographs, and drawings. The species hypothesis is supported by analysis of a 658 bp fragment of cytochrome c oxidase subunit I (COI). Divergences in the COI gene are evaluated using maximum likelihood and Bayesian inference analyses. The species delimitation is assessed using Assemble Species by Automatic Partitioning (ASAP) and Poisson Tree Processes (PTP) methods. Genetic distances are visualised using multidimensional scaling. Mordellistena platypoda Selnekovič, Goffová & Kodada, sp. nov. is recovered as a well-separated species by both molecular and morphological analyses. Our results show that M. platypoda Selnekovič, Goffová & Kodada, sp. nov. is most closely related to M. tarsata Mulsant, 1856, although the two species differ significantly in vestiture colouration, presence of lateral ctenidia on the third metatarsomere, and presence of sexual dimorphism on the protibia. The results indicate that such morphological differences, which were traditionally used to distinguish between species groups, may in fact be present between closely related species. Interestingly, examination of the numerous museum material did not reveal additional specimens of the new species, and therefore M. platypoda Selnekovič, Goffová & Kodada, sp. nov. is currently known only from the Italian island of Ischia.

The Kalkalpen National Park is situated in Upper Austria and contains more than 800 springs. The international importance of this Park is, from the perspective of nature conservation directives, highly significant (European Nature Reserve Natura 2000, recognised wetland of the Ramsar convention). In the current study, the hydrobioid fauna (‘spring snails’) of the Kalkalpen National Park was evaluated. These tiny snails are difficult to determine; however, their investigation is especially desirable, as several species are threatened and as they are important for water quality assessment. Snails collected in 39 selected springs were examined with classical morphological methods (shell and genital anatomy) and, subsequently, by DNA analysis. For this task, the DNA barcode, a partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene (length of the sequence 658-682 bp), was PCR amplified and sequenced. From 107 specimens, the DNA barcoding sequence could be obtained and compared with already existing DNA sequences. The (sub)endemic species Bythinella conica, Hauffenia kerschneri, Hauffenia wienerwaldensis and Belgrandiella aulaei could be clearly identified. For Bythiospeum nocki, despite the ambitious collecting effort, only empty shells were found in four springs (including the locus typicus spring) in the Park and its surroundings. The genus Bythinella was detected in 36 springs. From 25 of these localities, DNA barcodes could be created, which matches those of Bythinella conica (comparison data from ABOL). It is, therefore, concluded that the species occurs widely in the Kalkalpen National Park. The genus Hauffenia was sampled from 16 springs. From one, the haplotype of Hauffenia wienerwaldensis could be identified (spring is 5 km outside the Park) and from six, the haplotype of Hauffenia kerschneri. Belgrandiella aulaei was found in three springs, which all lie outside the boundaries and are, therefore, not included in the protection measures of the National Park. The data and analyses obtained contribute to the assessment of the taxonomic status of the species studied. The present study gives a good baseline for further monitoring of the hydrobioids in the Kalkalpen National Park, which is important to evaluate current as well as to decide on future protection measures for this group.

Using the genus Casearia, we assessed the status of nested singletons: individual specimens corresponding to accepted species but in molecular trees appearing nested within clades of closely related species. Normally, such cases would be left undecided, while on the other hand, timely taxonomic decisions are required. We argue that morphological, chorological, and ecological data can be informative to illuminate patterns of speciation. Their use can provide a first step in testing taxon concepts at species level. We focused on five cases of nested singletons in trees of the genus Casearia. We employed PCA and cluster analysis to assess phenotypic differentiation. Using geocoordinates, we calculated niche space differentiation based on 19 bioclim variables, by means of PCA and niche equivalency and similarity tests and generated dot maps. We found that the singletons were morphologically distinctive in two of the five cases (Casearia selloana and C. manausensis), relatively distinctive in two other cases (C. zizyphoides and C. mariquitensis), and partially overlapping in the last case (C. grandiflora). For two cases (C. mariquitensis and C. selloana), ecological niche space was broadly overlapping, in two cases it was found broadly nested (C. grandiflora and C. zizyphoides), and in one case narrowly nested (C. manausensis), but in no case niche differentiation was observed. Niche overlap, similarity and equivalency showed corresponding patterns. Given these data, one would interpret C. selloana and C. manausensis as presumably well-distinguished taxa, their narrow distribution ranges suggesting recently emerging lineages. The other three cases are not clearcut. Morphological data would suggest particularly C. grandiflora conspecific with C. arborea, but differences in the distribution are intriguing. Our approach would reject the notion of potential synonymy based on nested phylogenetic placement for at least two of the five cases. The other case also shows no complete lack of differentiation which would support synonymy.

Munidopsid squat lobsters are among the most abundant decapods at abyssal depths and the most diverse squat lobster group in the East Pacific region. During recent cruises along the East Pacific, many deep-sea squat lobsters were collected. Among these, we described five new munidopsid species supported both by morphological characters and molecular phylogenetics: Munidopsis girguisi sp. nov., M. nautilus sp. nov., M. testuda sp. nov., M. cortesi sp. nov. and M. hendrickxi sp. nov. We also report new records of several Munidopsis species across the East Pacific that increase the species distribution ranges. Here, we reconstructed the phylogenetic relationships of the East Pacific species in relation to other Galatheoidea using one nuclear and two mitochondrial gene fragment(s); we also performed single locus species delimitation analyses to explore the species status of various East Pacific munidopsid taxa. The new taxa were photographed, illustrated and imaged with micro-computed tomography. The phylogenetic results show that: (1) Janetogalathea californiensis, previously included in the family Galatheidae, nests within Munidopsidae; (2) the phylogenetic position of Phylladiorhynchus and Coralliogalathea as belonging in Galatheidae is not supported; and (3) Munidopsis is paraphyletic, agreeing with recent systematic hypotheses. Short genetic distances and species delimitation analyses suggested that a clade mostly constituted by abyssal species might include fewer species than currently considered, as species show a wider geographic range than previously considered, conforming with traditional hypotheses of cosmopolitanisms in abyssal species. ZooBank: urn:lsid:zoobank.org:pub:CED9EB18-7061-47A7-B2FF-7F1DAFCC7B12.

The Chromolaena congesta complex (Asteraceae, Eupatorieae) presents a difficult challenge for biodiversity researchers due to the intertwined connections of its species and the difficulty of establishing morphological boundaries. In this study, we aimed to use morphometric analyses to evaluate the delimitation of the taxa belonging to the C. congesta complex, identify informative morphological traits and to understand the identity of several “atypical” specimens morphologically related to the complex. To achieve this, we used cluster and principal component analysis to evaluate 50 morphological traits from a total of 210 specimens throughout the geographic distribution of the species complex. We found support for the recognition of at least six species - Chromolaena ascendens, C. gentianoides, C. hirsuta, C. latisquamulosa, C. rhinanthacea and C. squarrulosa - according to a phenetic species concept and to corroborate the restoration of three species from synonymy. Furthermore, our results provide a morphological recircumscription of C. congesta and C. elliptica and indicate two taxonomic novelties, including a new combination and a new species. Despite the answers provided by morphology, many taxonomic issues remain to be solved, and further studies with other types of evidence should be carried to contribute towards a more stable classification.

Microsatellite data used to produce genetic structuring (Fig. 2) and PCoA (Fig. S3) are accessible on the online UMR PVBMT Cirad dataverse (https://doi.org/10.18167/DVN1/3OFMLH).; International audience; The leafless Vanilla species complex from the South-West Indian Ocean (SWIO) region has long been a taxonomic challenge, due to limited patterns of morphological differentiation and an absence of variation within chloroplast sequences. This complex includes seven known morphospecies: V. madagascariensis, V. bosseri, V. decaryana, and V. perrieri endemic to Madagascar, V. humblotii presumed as endemic to the Comoros Archipelago, but also present in Madagascar, V. roscheri from the East African coast, and V. phalaenopsis endemic to Seychelles. A previous population genetic study using microsatellite markers allowed us to distinguish, in addition to the five recognized Malagasy taxa, two other genetic clusters present in the East of the island. An integrative taxonomy approach was therefore conducted by combining microsatellite and morphological data used in the previous study with new data sets, and by adding ITS sequencing data, to validate the taxonomic level of these Malagasy genetic clusters and unravel phylogenetic relationships between SWIO species. As a result, based on phylogenetic, genotypic and morphological evidence, nine species were discriminated in the SWIO region, including seven in Madagascar, with two new eastern species. The leafless Vanilla group originated and diversified in Madagascar, from an ancestor of African descent, with three subsequent independent colonization events from Madagascar to the other territories of SWIO within the two main lineages (white versus yellow flower species). The new Malagasy species, V. allorgeae Andriamihaja & Pailler sp. nov., and V. atsinananensis Andriamihaja & Pailler sp. nov., are described and a new identification key is proposed.

One new species of the genus Dendronotus (Nudibranchia: Dendronotidae) is described from Norway and Northern Ireland, as well as from the adjacent North Sea, and one new subspecies of Dendronotus arcticus is described from Norway by applying a combination of fine-scale morphological and molecular phylogenetic data. The present case demonstrates multilevel morphological and molecular similarities and differences considering on the one hand a grouping of three similar looking sympatric taxa (D. yrjargul, D. arcticus gartensis n. subsp. and D. keatleyae n. sp.), and on the other hand two different looking apparently allopatric subspecies (D. arcticus arcticus and D. arcticus gartensis n. subsp.). The type species of the genus, D. frondosus, which is the commonest dendronotid in Norway and the United Kingdom, consistently demonstrates substantial molecular and fine-scale morphological differences from D. keatleyae n. sp. The present study, apart from providing purely taxonomic information, also provides new data for a broad discussion of global biodiversity patterns. Similar Ones Are Not Related and Vice Versa—New Dendronotus Taxa (Nudibranchia: Dendronotidae) from the North Atlantic Ocean Provide a Platform for Discussion of Global Marine Biodiversity Patterns

In Europe, the V. epipsila—V. palustris group comprises V. epipsila Ledeb., V. palustris L., V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta), interspecific hybrids, and putative introgressants. The genetic affinity of V. pubifolia to V. palustris, and their shared origin via hybridization followed by polyploidization, were confirmed using inter simple sequence repeat (ISSR) markers, restriction site-associated DNA sequencing (RAD-Seq), and a low-copy nuclear gene, GPI, which encodes glucose-6-phosphate isomerase. The other taxa of subsect. Stolonosae were not identified as putative parents of V. pubifolia by GPI. Our analyses indicated that V. pubifolia can be included in the morphological and genetic variation of V. palustris. The ISSR, RAD-Seq, and genome size value separated well V. palustris from V. epipsila and hybrids. The results also reopen the discussion on intraspecific variation in the context of taxa ranks and species concepts. The reduced tolerance of V. epipsila in Europe to changing environmental conditions might result from low genetic differentiation and heterozygosity, as well as the increased number of interspecific hybrids (V. epipsila × V. palustris), and eventually can possibly lead to its extinction. The disappearance of populations/individuals of this species may indicate anthropogenic changes occurring in peatlands.

Sri Lanka is a local hotspot for Cnemaspis day geckos with 40 currently known species with 100% endemism. In this paper, we evaluate the phylogenetic relationships of Cnemaspis species belonging to the alwisi group of the podihuna clade and describe two additional new species of Cnemaspis from Sri Lanka; one from Galgiriya mountain, Kurunegala District, and another from Ethagala mountain, Ampara District. These new species were recorded from granite caves within forested areas in isolated mountains in the dry bioclimatic zone (point-endemics). Both new species are microhabitat specialists with narrow niches limited to humid, cool, canopy-shaded granite caves and old buildings associated with granite caves, where they are camouflaged by their cryptic morphology and body colouration. Furthermore, both species prefer narrow (~ 6–12 mm), long (~ 120–450 mm) and deep (~ 80–260 mm) crevices as refugia. The regions in which these habitats are located receive relatively low annual rainfall (1,000–1,500 mm). These new species are medium in size (28.5–36.8 mmSVL) and can be differentiated from all other Sri Lankan Cnemaspis by the presence of clearly enlarged, subhexagonal subcaudal scales and the absence of precloacal pores in males. Both species described here are categorised herein as Critically Endangered (CR) under the IUCN Red List criteria. The major threats for these new species are habitat loss due to expansion of commercial-scale agriculture, illicit forest encroachments, and forest fires. Therefore, we recommend that relevant authorities take immediate conservation action to ensure the protection of these forest areas with their buffer zones in the near future.

The Reithrodontomys tenuirostris species group is considered “the most specialized” within the genus Reithrodontomys from morphological and ecological perspectives. Previous studies based on molecular data recommended changes in the taxonomy of the group. In particular, R. microdon has been the most taxonomically questioned, with the suggestion that it constitutes a complex of cryptic species. We analyzed the phylogenetic relationships of the R. tenuirostris species group using DNA sequences from the mitochondrial Cytochrome b gene and Intron 7 of the nuclear beta fibrinogen gene. In addition, divergence times were estimated, and possible new taxa delimited with three widely used species delimitation methods. Finally, possible connectivity routes based on shared haplotypes were tested among the R. microdon populations. All species were recovered as monophyletic with the exception of R. microdon, whose individuals were grouped into four different haplogroups, one of which included specimens of R. bakeri. Diversification within the R. tenuirostris species group began about 3 Ma, in the Pleistocene. The bGMYC and STACEY delimitation methods were congruent with each other, delimiting at the species-level each haplogroup within R. microdon, while the mPTP suggested a greater number of species. Moreover, none of the haplogroups showed potential connectivity routes between them, evidencing lack of gene flow. Our results suggest the existence of a higher number of species in the R. tenuirostris group, because we show that there are four species within what is currently recognized as R. microdon.

Ficus krishnae is considered as native to India and is well-known for the peculiarity in nature of its cup-shaped leaves where both the vernacular name (Krishna Fig) and specific epithet were derived. The taxonomic status of Ficus krishnae is still unclear and currently treated as a subspecies or variety under Ficus benghalensis. In the present study, morphological characters and molecular analysis were employed to address their species delimitation. The spacer markers ITS2 and trnH-psbA were used for constructing phylogenetic trees along with morphometric analysis. Ficus krishnae distinctly differs from Ficus benghalensis by having cup-forming leaves and the nature of the aerial roots, stipules, petioles, ostiolar bracts of the receptacle, DNA content, chromosome differences and nodal anatomy. The results showed that the highest divergence is observed in trnH-psbA (20.8 ± 12.2), followed by ITS2 (5.7 ± 3.2). The phylogenetic tree construction using Bayesian analysis showed a divergent boundary between the two species suggesting that F. krishnae could be an independent species, not a variety of F. benghalensis. The present study’s findings support the view that these two floras can be treated as different species.

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories., We investigate species boundaries and the potential for micro‐endemism for a charismatic genus of lichen fungi occurring in Baja California. Genome‐scale data support high species‐level diversity, along with high levels of endemism, while highlighting the problem of delimiting species, particularly in groups with complex, recent phylogeographic histories.

Niphargus enslini Karaman, 1932 was collected only once in 1905 from the Falkensteiner Höhle (Baden-Württemberg, Germany). Two years after its description, the species was synonymized with Niphargus virei and not studied any more. During recent surveys on German niphargids, further samples collected in this cave did not yield N. enslini specimens, but this species was collected in the Blätterteighöhle and in the Schwarzer Brunnen, two caves located in Baden-Württemberg and intercepting the same karstic aquifer feeding Falkensteiner Höhle. In an integrative taxonomic approach, we carefully studied the morphology of the newly collected specimens and sequenced two molecular markers (fragments of the cytochrome c oxidase subunit I (COI) and of the nuclear 28S rRNA gene) to test for possible conspecificity of N. enslini with N. virei. Morphological analysis confirmed that N. enslini is distinct from the N. virei species complex. We provide a redescription of newly collected material, together with new drawings of a more than 100 years old topotypic female. We briefly discuss the putative origin of N. enslini and the age of its split from the N. virei species complex.

Abstract The genus Calotes Cuvier, 1817 (Agamidae: Draconinae) is highly diverse, with species occurring in South and Southeast Asia, and Oceania. Most species of the subfamily except C. versicolor have narrow geographic distributions. Calotes versicolor is distributed from western Iran in the west to south China and Indonesia in the east and has been introduced to parts of Africa and North America. The species has had a complicated taxonomic history; multiple species and subspecies related to C. versicolor were described from India and adjoining regions, which were synonymized in subsequent revisions. However, a study of Burmese C. versicolor yielded two new species, C. htunwini and C. irawadi, indicating that C. versicolor is a species complex. Such integrative taxonomic studies have not been carried out in India, the supposed type locality of C. versicolor. Hence, we studied C. versicolor sensu lato from the Indian subcontinent and generated sequences of mitochondrial 16S and COI fragments from tissues sampled from multiple localities in the region, including the type localities of its synonyms. Phylogenetic analyses revealed four well-supported, deeply-divergent lineages, supported by morphological data. These lineages represent (i) C. versicolor sensu stricto, from South India and parts of the east coast, (ii) C. irawadi sensu lato from northeast India and Southeast Asia, (iii) a synonym from the eastern Indo-Gangetic Plains which we resurrect here, and (iv) a subspecies from Pakistan which we elevate to species level. We provide re-descriptions for the resurrected or elevated species, and a diagnostic key to the species of the C. versicolor complex. The study shows that C. versicolor sensu stricto is endemic to parts of southern and eastern India, and not widely distributed, though it may have been introduced to other parts of the world.

The diving beetle genusPeschetiusGuignot, 1942 (Coleoptera: Dytiscidae) in India is reviewed. Integrative taxonomic approach using morphology, multivariate morphometry and genetic analysis of cytochrome oxidase subunit 1 revealed the presence of four species, two of which are described here as new:Peschetius bistroemisp. nov.from southern Western Ghats (Kerala) differs from all known congeners with distinctly broadened male antennomeres IV and V, shape of the prosternal process and the male genitalia;P. nilssonisp. nov.from northern Western Ghats, Rajasthan and Madhya Pradesh is similar to the widespread IndianP. toxophorusGuignot, 1942, from which it differs in habitus, elytral colour pattern and the shape of the male genitalia. New records are presented for the remaining Indian species, namelyP. quadricostatus(Aubé, 1838) andP. toxophorus. All species are diagnosed, illustrated and a key to their identification is provided.

Phylogenetic relationsships of four nominal Lucanus Scopoli, 1763 species, L. swinhoei Parry, 1874, L. continentalis Zilioli, 1998, L. liuyei Huang & Chen, 2010, and L. wuyishanensis Schenk, 1999, are assessed based on mitochondrial (16S rDNA, COI) and nuclear (28S rDNA, Wingless) genes. The genetic distance is 0.0072 between L. swinhoei and L. continentalis, and 0.0094 between L. wuyishanensis and L. liuyei. Three species-delimitation approaches (ABGD, PTP, and GMYC) consistently showed L. swinhoei + L. continentalis and L. wuyishanensis + L. liuyei as two MOTUs. A new synonymy, L. liuyei = L. wuyishanensis, is proposed. Synonymy of L. swinhoei over L. continentalis is confirmed.

Mites of the genus Geckobia (Acariformes: Pterygosomatidae) are permanent and highly specialised ectoparasites of geckos (Gekkota). We conducted a local study on Geckobia mites associated with the geckos of the family Gekkonidae found mainly in the territory of the Indian Institute of Science’s campus (Bangalore, India). In total, we examined 208 lizards belonging to two genera: Hemidactylus and Cnemaspis. We assessed the prevalence of the mites and identified the preferred site for their infestation. We extended the standard morphological identification of the mite species by using DNA barcode markers, partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and nuclear ribosomal gene sequences: 18S rRNA and hypervariable region D2 of nuclear 28S rRNA. We checked the suitability of COI and nuclear (D2 of 28S rRNA) markers for species delimitations and identification purposes of the genus. The distance- and phylogeny-based approaches were applied: (i) to test the presence of a barcoding gap, we used the automated barcoding gap discovery tool (ABGD) and investigated intra- and interspecific genetic distances, and (ii) to reconstruct evolutionary relationships within the species, we performed maximum likelihood (ML) and Bayesian inference with Markov-Chain Monte Carlo (BI) analyses. As a result, we described five new species—Geckobia gigantea sp. n., G. treutleri sp. n., G. unica sp. n. and G. brevicephala sp. n.—from four Hemidactylus species: H. giganteus, H. treutleri, H. parvimaculatus and H. frenatus, respectively, and G. mysoriensis sp. n. from Cnemaspis mysoriensis. Additionally, we found three already described species: Geckobia indica Hirst, 1917 on H. treutleri (new host), Geckobia bataviensis Vitzhum, 1926 on H. parvimaculatus (new host) and H. frenatus (new locality) and Geckobia phillipinensis Lawrence, 1953 on H. frenatus (new locality). The diagnoses of G. indica and G. phillipinensis were improved and supplemented by descriptions of the males and juveniles. Both topologies of the BI and ML phylogenetic trees, as well as genetic distances, supported the species boundaries in the mite population shown by the morphological data. Hemidactylus frenatus was the most infected gecko species (61% prevalence), with the highest number of mite species (three spp.). The scale-mite richness was higher than expected; therefore, further research is required to evaluate the true diversity of Geckobia mites.

Ideally, the footprint of the evolutionary history of a species is drawn from integrative studies including quantitative and qualitative taxonomy, biogeography, ecology, and molecular genetics. In today’s research, species delimitations and identification of conservation units is often accompanied by a set of—at minimum—two sequence markers appropriate for the systematic level under investigation. Two such studies re-evaluated the species status in the world’s largest Odonata, the Neotropical damselfly Megaloprepus caerulatus. The species status of the genus Megaloprepus has long been debated. Despite applying a highly similar set of sequence markers, the two studies reached different conclusions concerning species status and population genetic relationships. In this study, we took the unique opportunity to compare the two datasets and analyzed the reasons for those incongruences. The two DNA sequence markers used (16S rDNA and CO1) were re-aligned using a strict conservative approach and the analyses used in both studies were repeated. Going step by step back to the first line of data handling, we show that a high number of unresolved characters in the sequence alignments as well as internal gaps are responsible for the different outcomes in terms of species delimitations and population genetic relationships. Overall, this study shows that high quality raw sequence data are an indispensable requirement, not only in odonate research.

The deep-sea holothurian Chiridota heheva Pawson & Vance, 2004 was recently recognised as a cosmopolitan species which exploits the organic enrichment at three types of deep-sea reducing environments: hydrothermal vents, cold seeps, and organic falls. Here, we apply phylogenetic reconstruction and species delimitation approaches using new COI and 12S sequence data to show that C. heheva is genetically congruent with the only other hydrothermal vent holothurian, Chiridota hydrothermica Smirnov & Gebruk, 2000, with strong supporting morphological, ecological, and biogeographical parallels between the two. As such, we propose that C. heheva is a junior synonym of C. hydrothermica, and that subsequent chiridotid holothurians discovered at deep-sea reducing environments likely also belong to this single globally distributed species. As a species endemic to deep-sea reducing environments yet known at sites across the Atlantic, Indian, and Pacific Oceans, this unique holothurian provides an interesting case study for connectivity, biogeography, and speciation in the deep sea, which has important implications for deep-sea conservation planning.

Accurate species delimitation is critical for biodiversity conservation. Integrative taxonomy has been advocated for a long time, yet tools allowing true integration of genetic and phenotypic data have been developed quite recently and applied to few models, especially in plants. In this study, we investigated species boundaries within a group of twelve Pseudophrys taxa from France by analyzing genetic, morphometric and chemical (i.e., floral scents) data in a Bayesian framework using the program integrated Bayesian Phylogenetics and Phylogeography (iBPP). We found that these twelve taxa were merged into four species when only genetic data were used, while most formally described species were recognized as such when only phenotypic (either morphometric or chemical) data were used. The result of the iBPP analysis performed on both genetic and phenotypic data supports the proposal to merge Ophrys bilunulata and O. marmorata on the one hand, and O. funerea and O. zonata on the other hand. Our results show that phenotypic data are particularly informative in the section Pseudophrys and that their integration in a model-based method significantly improves the accuracy of species delimitation. We are convinced that the integrative taxonomic approach proposed in this study holds great promise to conduct taxonomic revisions in other orchid groups.

Elopidae is the most speciose family within the Elopiformes, comprising seven valid species. Despite this reduced number of species, the family presents poorly resolved systematics, mainly owing to its wide distribution and highly conserved anatomic features. Therefore, we aimed to explore the species diversity of the Elopidae using species delimitation, genetic diversity, and phylogenetic analysis combined with DNA barcoding of the COI gene. The results from the delimitation analysis grouped the species into a single cluster, while the genetic diversity analysis among the groups showed a distance ranging between 1.29 and 2.78%. Both phylogenetic and haplotype network analysis grouped the species into four clades, associated with the distribution of the organisms. The lack of resolution in the species delimitation analysis might be directly associated with the recent radiation of the group, a hypothesis corroborated by both the low genetic diversity (close to the 2% threshold) and the few mutations that separate the haplotypes observed among the species. Interestingly, our data supported a new arrangement for the Elops species. In addition, the data available in public databases present taxonomic errors at several levels. Although some issues remain unsolved, our results can be used in the identification of taxa and provide information to assist taxonomic revisions of the Elopidae.

Supplemental material for this article can be accessed here: https://doi.org/10.1080/14772000.2022.2130464; International audience; Determining the relative importance of dispersal and vicariance events across neotropical regions is a major goal in biogeography. These events are thought to be related to important landscape changes, notably the transition of Amazonia toward its modern hydrological configuration ca. 10 million years ago. We investigated the spatio-temporal context of the diversification of one of the major lineages of Pristimantis, a widespread and large genus of direct-developing Neotropical frogs. We gathered a spatially and taxonomically extensive sampling of mitochondrial DNA sequences from 754 Pristimantis gr. conspicillatus specimens, which led to delimiting 75 Operational Taxonomic Units (OTUs). Complete mitogenomes of 35 of these OTUs were assembled and collated with two nuDNA loci to reconstruct a time-calibrated phylogeny. We identified five major clades that diverged around the Oligocene-Miocene transition and that are largely restricted to distinct Neotropical regions i.e. Western Amazonia (P. conspicillatus clade), the Brazilian Shield (P. fenestratus clade), the Atlantic Forest (P. ramagii clade), the Guiana Shield (P. vilarsi clade) and the northern Andes (P. nicefori clade). The majority of the diversification events within these clades occurred in-situ from the early Miocene onward. Yet, a few ancient dispersal/vicariance events are inferred to have occurred among trans-Andean forests, the Atlantic Forest, the Brazilian and the Guiana Shields, but almost none in the last 10 Ma. The radical landscape transformations during the Miocene caused by the Andean orogeny and hydrological barriers such as the Pebas System and the subsequent transcontinental configuration of the Amazon drainage is a likely explanation for the isolation of the different clades within the P. gr. conspicillatus.

In the context of diminishing global biodiversity, the validity and practicality of species delimitation methods for the identification of many neglected and undescribed biodiverse species have been paid increasing attention. DNA sequence-based species delimitation methods are mainly classified into two categories, namely, distance-based and tree-based methods, and have been widely adopted in many studies. In the present study, we performed three distance-based (ad hoc threshold, ABGD, and ASAP) and four tree-based (sGMYC, mGMYC, PTP, and mPTP) analyses based on Trochoidea COI data and analyzed the discordance between them. Moreover, we also observed the performance of these methods at different taxonomic ranks (the genus, subfamily, and family ranks). The results suggested that the distance-based approach is generally superior to the tree-based approach, with the ASAP method being the most efficient. In terms of phylogenetic methods, the single threshold version performed better than the multiple threshold version of GMYC, and PTP showed higher efficiency than mPTP in delimiting species. Additionally, GMYC was found to be significantly influenced by taxonomic rank, showing poorer efficiency in datasets at the genus level than at higher levels. Finally, our results highlighted that cryptic diversity within Trochoidea (Mollusca: Vetigastropoda) might be underestimated, which provides quantitative evidence for excavating the cryptic lineages of these species.

Cambeva contains species with complex taxonomy or poorly delimitated in terms of morphology and geopraphic distribution. We conducted an extensive review of Cambeva populations from coastal drainages of Southern to Southeastern Brazil to evaluate species geographic limits with an integrative analysis including morphological and molecular data (COI). We test if two single-locus methods, Bayesian Poisson Tree Processes (bPTP) and Generalized Mixed Yule Coalescent (GMYC), are efficient to delimit species boundaries in Cambeva by the comparison with the diagnosable morphological units. Using GMYC, we also evaluated the combination of tree and molecular clock priors to reconstruct the input phylogeny and assessed how well the implemented model fitted our empirical data. Eleven species were identified using a morphological diagnosability criterion: Cambeva balios, C. barbosae, C. botuvera, C. cubataonis, C. davisi, C. guaraquessaba, C. iheringi, C. tupinamba, and C. zonata and two treated as undescribed species. In contrast with previous knowledge, many of them have wider distribution and high intraspecific variation. Species delimitation based on single-locus demonstrated incongruences between the methods and strongly differed from the morphological delimitation. These disagreements and the violation of the GMYC model suggest that a single-locus data is insufficient to delimit Cambeva species and the failure may be attributable to events of mitochondrial introgression and incomplete lineage sorting. Resumo Cambeva contém espécies com taxonomia complexa ou mal delimitadas em termos morfológicos e de distribuição geográfica. Realizamos uma extensa revisão de populações de Cambeva das drenagens costeiras do Sul ao Sudeste do Brasil para avaliar os limites das espécies com uma análise integrativa incluindo dados morfológicos e moleculares (COI). Testamos se dois métodos de locus único, Implementação Bayesiana dos Processos da Árvore de Poisson (bPTP) e Coalescente de Yule Misto Generalizado (GMYC), são eficientes para delimitar os limites das espécies em Cambeva pela comparação com as unidades morfológicas diagnosticáveis. Usando o GMYC, também avaliamos a combinação de árvores e relógios moleculares para reconstruir a filogenia e avaliamos o quão bem o modelo implementado se ajustava aos nossos dados empíricos. Foram identificadas 11 espécies usando o critério morfológico: Cambeva balios, C. barbosae, C. botuvera, C. cubataonis, C. davisi, C. guaraquessaba, C. iheringi, C. tupinamba e C. zonata e duas tratadas como espécies não-descritas. Em contraste com o conhecimento prévio, muitas delas têm distribuição mais ampla e alta variação intraespecífica. A delimitação das espécies baseada em locus único demonstrou incongruências entre os métodos e diferiu fortemente da delimitação morfológica. Essas discordâncias e a violação do modelo GMYC sugerem que os dados de locus único são insuficientes para delimitar as espécies de Cambeva e a falha pode ser atribuída a eventos de introgressão mitocondrial e sorteio incompleto da linhagem.

Environmental DNA metabarcoding is becoming a predominant tool in biodiversity assessment, as this time- and cost-efficient tactics have the ability to increase monitoring accuracy. As a worldwide distributed genus, Rheocricotopus Brundin, 1956 still does not possess a complete and comprehensive global DNA barcode reference library for biodiversity monitoring. In the present study, we compiled a cytochrome c oxidase subunit 1 (COI) DNA barcode library of Rheocricotopus with 434 barcodes around the world, including 121 newly generated DNA barcodes of 32 morphospecies and 313 public barcodes. Automatic Barcode Gap Discovery (ABGD) was applied on the 434 COI barcodes to provide a comparison between the operational taxonomic units (OTU) number calculated from the Barcode Index Number (BIN) with the "Barcode Gap Analysis" and neighbor-joining (NJ) tree analysis. Consequently, these 434 COI barcodes were clustered into 78 BINs, including 42 new BINs. ABGD yielded 51 OTUs with a prior intraspecific divergence of Pmax = 7.17%, while NJ tree revealed 52 well-separated clades. Conservatively, 14 unknown species and one potential synonym were uncovered with reference to COI DNA barcodes. Besides, based on our ecological analysis, we discovered that annual mean temperature and annual precipitation could be considered as key factors associated with distribution of certain members from this genus. Our global DNA barcode reference library of Rheocricotopus provides one fundamental database for accurate species delimitation in Chironomidae taxonomy and facilitates the biodiversity monitoring of aquatic biota., Ecology and Evolution, 11 (17), ISSN:2045-7758

With a Nearctic distribution, the family Cambaridae harbors a high species richness in Mexico, which is also evident along the Pánuco River catchment. A series of surveys carried on in five populations from the Sierra Gorda Biosphere Reserve in the State of Querétaro resulted in localizing a putative new species for science. A molecular phylogenetic study and species delimitation analyses including all the known Procambarus species from the Pánuco River catchment were conducted based on three mitochondrial genes (16S rDNA, 12S rDNA, and COI; 2,462 bp in total). Phylogeny recovered all species as monophyletic, including the populations under study. All delimitation results based on barcoding, ABGD, GMYC, bPTP, and gonopod differentiation agree in the recognition of a new taxon, to which the name Procambarus xihuisp. nov. is given, and its diagnosis and description are provided. The new species can be distinguished from the remaining species in the genus, among other characters, by a unique configuration of the terminal elements of the first pleopod of form I male, which includes a central projection lamellate, hood-like, forming a concave blade-like structure mesially directed, as well as a caudal process crest-like, mesiodistally directed, forming a lateral side of the concavity.

Lactifluus (Pers.) Roussel is an ectomycorrhizal genus that was recently recognized to be distinct from the genus Lactarius. To date, 216 Lactifluus species have been reported worldwide. Misidentification of Lactifluus species is common because of intraspecific morphological variation, cryptic diversity, and the limited number of taxonomic keys available. Molecular data are indispensable for species delimitation; a multilocus phylogenetic analysis showed that most Asian Lactifluus species are not conspecific with morphologically similar species present on other continents. In particular, Korea has misused European and North American Lactifluus names. In this study, we evaluated the taxonomy of Lactifluus in Korea using both morphological and multilocus molecular (ITS, nrLSU, rpb1, and rpb2) data. We examined 199 Lactifluus specimens collected between 1980 and 2016, and a total of 24 species across the four Lactifluus subgenera were identified. All Korean species are distinct and clearly separated from European and North American species. Five taxa corresponded to previously described species from Asia and the remaining 19 taxa are confirmed as new species. Herein, we provide keys to the Korean Lactifluus species within their subgenera, molecular phylogenies, a summary of diversity, and detailed description of the new species.